SATB2
The gene encoding special AT-rich sequence-binding protein 2 (SATB2) was identified in 1999 during the massive effort of sequencing the human genome (Kikuno R et al (1999) DNA Res. 6:197-205). Since then, the SATB2 gene has been considered as expressed mainly in neuronal tissue.
SATB2 is a transcription factor that form parts of the nuclear matrix and orchestrates gene expression in a tissue-specific manner by regulating high-order chromatin structure through interaction with AT-rich sequences, also referred to as matrix attachment regions (MARs) (Dickinson L A et al (1992) Cell 70, 631-45; FitzPatrick D R et al (2003) Hum. Mol. Genet. 12, 2491-501; Yasui D, (2002) Nature 419, 641-5; Bode, J (2000) Crit. Rev. Eukaryot. Gene. Expr. 10, 73-90).
Studies of the gene and its protein product, the SATB2 protein, point towards an involvement in regulation of gene expression as a transcription factor in neuronal tissue (Dobreva G et al (2003) Genes Dev. 17:3048-3061; Britanova O et al (2005) Eur. J. Neurosci. 21:658-668). The SATB2 gene has also been described to have a role in palate development and cleft palate (FitzPatrick D R et al (2003) Human Mol. Genet. 12:2491-2501; van Buggenhout G et al (2005) Eur. J. Med. Genet. 48:276-289).
Salahshor et al studied a patient with the adenomatous polyopsis coli (APC) gene mutation (Salahshor et al (2005) BMC cancer 5:66). APC patients develop an abnormal amount of colonic adenomas at a young age that eventually, if left untreated, will progress to colo-rectal cancer. Global gene expression profiling revealed that a group of 84 genes, including SATB2, had a significantly altered expression in adenomas compared to normal mucosa. SATB2 was found significantly down-regulated but was not selected for any further analysis. A recent expression profiling study of colo-rectal cancer in Int J Cancer likewise indicated an altered expression status for SATB2 at the mRNA level (Groene J et al (2006) Int J Cancer 119, 1829-1836).
PCT publications WO03/022126 and WO2006/015742 describe other, similar studies directed to expression profiling of cancer cells. The expression of a multitude of genes, including SATB2, is analyzed and conclusions are drawn from the overall expression patterns.
Importantly, the studies referred to above provide no suggestions concerning the use of the SATB2 protein as a specific colo-rectal marker or the use of SATB2 as a prognostic tool for colo-rectal cancer.
Cancer
Cancer is one of the most common causes of disease and death in the western world. In general, incidence rates increase with age for most forms of cancer. As human populations continue to live longer, due to an increase of the general health status, cancer will affect an increasing number of individuals. The cause of most common cancer types is still at large unknown, although there is an increasing body of knowledge providing a link between environmental factors (dietary, tobacco smoke, UV radiation etc) as well as genetic factors (germ line mutations in “cancer genes” such as p53, APC, BRCA1, XP etc) and the risk for development of cancer.
No definition of cancer is entirely satisfactory from a cell biological point of view, despite the fact that cancer is essentially a cellular disease and defined as a transformed cell population with net cell growth and anti-social behavior. Malignant transformation represents the transition to a malignant phenotype based on irreversible genetic alterations. Although this has not been formally proven, malignant transformation is believed to take place in one cell, from which a subsequently developed tumor originates (the “clonality of cancer” dogma). Carcinogenesis is the process by which cancer is generated and is generally accepted to include multiple events which ultimately lead to growth of a malignant tumor. This multi-step process includes several rate-limiting steps, such as addition of mutations and possibly also epigenetic events, leading to formation of cancer following stages of precancerous proliferation. The most common forms of cancer arise in somatic cells and are predominantly of epithelial origin (skin, prostate, breast, colon and lung) followed by cancers originating from the hematopoetic lineage (leukemia and lymphoma) and mesenchymal cells (sarcomas). The stepwise changes involve accumulation of errors (mutations) in vital regulatory pathways that determine cell division, asocial behavior and cell death. Each of these changes provides a selective Darwinian growth advantage compared to surrounding cells, resulting in a net growth of the tumor cell population. It is important to emphasize that a malignant tumor does not only consist of the transformed tumor cells themselves but also surrounding normal cells which act as a supportive stroma. This recruited cancer stroma consists of connective tissue, blood vessels and various other normal cells, e.g. inflammatory cells, which act in concert to supply the transformed tumor cells with signals necessary for continued tumor growth.
Cancer Diagnostics
Microscopic evaluation of a tissue section taken from a tumor remains the golden standard for determining a diagnosis of cancer. Analysis of genomic DNA, transcribed genes and expressed proteins all add important information to the histological features detected in the microscope. Tomorrow's diagnosis, prognostic information and choice of treatment will in all likelihood be based on a synoptic evaluation of morphology in conjunction with analyses of nucleic acids and proteins. Already today, evolving knowledge based on the human genome sequence and biochemical pathways, including signaling inside and between cells in a tissue, enable the dissection of some of the mechanisms that underlie different stages in tumor formation as well as variation of phenotypes, which define the different types of cancer.
Despite remarkable progress within molecular biology, cancer diagnostics still relies on the use of light microscopy. The development of molecular tools has played an important, although as of yet incremental, role to discriminate a cancer cell from a normal cell. The most commonly used method in addition to histochemical staining of tissue sections is immunohistochemistry. Immunohistochemistry allows the detection of protein expression patterns in tissues and cells using specific antibodies. The use of immunohistochemistry in clinical diagnostics has provided a possibility to not only analyze tissue architecture and cellular morphology, but also to detect immunoreactivity in different cell populations. This has been important to support accurate grading and classification of different primary tumors as well as in the diagnostics of metastases of unknown origin. The most commonly used antibodies in clinical practice today include antibodies against cell type markers, e.g. PSA, MelanA, Thyroglobulin and antibodies recognizing intermediate filaments, cluster of differentiation (CD) antigens etc. and markers of malignant potential, e.g. Ki67, p53, HER-2. Aside from immunohistochemistry, the use of in situ hybridization for detecting gene amplification and gene sequencing for mutation analysis are evolving technologies within cancer diagnostics.
Colo-Rectal Cancer
Colo-rectal cancer is one of the most common forms of human cancer worldwide. Data from the GLOBOCAM 2002 database presented by Parkin et al show that around 1 million new cases of colo-rectal cancer are found yearly (Parkin et al (2007) CA Cancer J Clin 55, 74-108). Further, the incidence of colo-rectal cancer in the world is approximately 9.4% of all cancers, and colo-rectal cancer constitutes the second most common cause of death in the western world. The five-year survival rate of colo-rectal cancer is approximately 60% in the western world but as low as 30% in Eastern Europe and India.
Early detection and surgery with excision of the tumor is currently of critical importance for a favorable prognosis. Symptoms depend on where in the distal gastro-intestinal tract the tumor is located, and include bowel distress, diarrhea, constipation, pain and anemia (secondary to bleeding from the tumor into the bowel). Malignant tumors may be categorized into several stages according to different classification schemes, such as the TNM/UICC classification I-IV or Dukes' stages A-C. The least malignant tumors (Dukes' stages A and B) have a reasonably favorable outcome, while on the other end some highly malignant tumors with metastasis (Dukes' stage C) have poor survival rates. Current diagnostics are based on patient history, clinical and endoscopic examination (rectoscopy and colonoscopy) optionally followed by radiological mapping to determine extensiveness of tumor growth. In conjunction with endoscopic examination, tissue biopsies are performed from dubious lesions.
For microscopic diagnosis, biopsy material from suspected tumors is collected and examined under a microscope. To obtain a firm diagnosis, the tumor tissue is then fixated in formalin, histo-processed and paraffin embedded. From the resulting paraffin block, tissue sections can be produced and stained using both histochemical and immunohistochemical methods.
For localized tumors, i.e. tumors that have not evolved into a metastasizing disease, surgical intervention with radical resection of the tumor and surrounding bowel and tissues is performed. The surgical specimen is then sent to pathology for gross and microscopical analysis. This analysis forms the basis for staging of the tumor. The by far most common form of colo-rectal cancer is adenocarcinoma, representing a tumor of glandular origin, which can be highly, moderately or lowly differentiated.
For primary tumors, hematoxylin-eosin stained tissue sections are sufficient to enable a correct diagnosis and classification according to the different colo-rectal cancer classifications. However, as colo-rectal cancer is very common and has often grown to a considerable size before detection, metastases are not uncommon. The tumor typically metastasizes to regional lymph nodes, but distant metastasis in the liver and lung is not unusual. A common clinical problem with cancer is patients that present a metastasis of unknown origin. In the case where a metastasis is an adenocarcinoma, several possible primary tumors can be suspected, e.g. breast, prostate, pancreatic, stomach and colo-rectal cancer. For differential diagnostics, immunohistochemical markers can be used that recognize features inherent in the cell of origin. At present, cytokeratin 20 (CK20), an intermediate filament marker abundant in the glandular cells of the GI-tract, is used to characterize colo-rectal cancer. However, several other adenocarcinomas can also be positive for CK20 antibodies, whereas not all colo-rectal cancers are positive. Furthermore, there are no markers available today that can distinguish tumors of low malignancy grade and low risk for metastasis from highly malignant tumors with a reduced chance of survival.
In order for doctors to give specific treatment for the right type of cancer and as early as possible, the provision of new molecular markers that are specific to colo-rectal cancer alone, and affords the possibility of differentiating patients into different risk categories is crucial. In summary, there is a great demand for new means to advance the diagnostics and screening of colo-rectal cancer.